This supplemental application seeks to extend studies to determine the molecular processes underlying spontaneous Ca2+ release in smooth muscle cells. Spontaneous and triggered release of Ca2+ ions from the sarcoplasmic reticulum of smooth muscle cells is a key event associated with contraction of the smooth muscle and bronchoconstriction of the airways. In recent years it has become clear that this Ca2+ release process is quite complicated, with two major intracellular Ca2+ release. Ryanodine receptors comprise one family of Ca2+ release channels; the function of these channels in skeletal and cardiac muscle is well established, but their role in the contraction of smooth muscle is less clear. A prominent feature of the electrical activity of smooth muscle cells in the airways is the appearance of spontaneous depolarizing and spontaneous hyperpolarizing currents. These currents arise from the opening of calcium-sensitive membrane channels following a brief release of calcium from the sarcoplasmic reticulum through ryanodine receptors. To determine the underlying structures and channels mediating and regulating spontaneous Ca2+ release, we will conditionally inactivate ryanodine receptor type 2 (RYR2) in transgenic mice and utilize a novel Ca2+ sensing mouse developed in this laboratory. A transgenic mouse expressing a ligand activated Cre recombinase/estrogen receptor fusion protein under control of the smooth muscle specific myosin heavy chain promoter will be created and used to inactivate RYR2 in smooth muscle in adult mice. We will also develop the next generation of Ca2+ sensing mice that express a myosin light chain kinase/circularly permutated green fluorescent protein/calmodulin fusion protein that in smooth muscle. We have engineered mice expressing this protein in smooth muscle, which is a significant experimental advantage for the determination of Ca2+ signaling mechanisms in airways in vivo. These studies will further advance our understanding of the processes regulating airway tone in health and disease. [unreadable] [unreadable]